| Bacillus thuringiensis (Bt) is one of the well-studied and the most widely used insecticidal microorganism, for its nontoxic to human, animals and environment. Bt was widely used in biological controls of agriculture, forest and health pests. Bt cry8genes, which were toxic to Coleoptera insect pests (such as Scarabaeidae, Curculionidae, Chrysomelidae pests, et al.), had greatly potential application value. To further exploit abundant resource of Bt in our country, work was done to screen Bt strains with high effective and broad-spectrum insecticidal activity and clone more cry8genes with toxicity to Coleoptera and independent intellectual property rights. All of the work had important significance, providing not only effective gene resources to cultivation transgenic plants, but also the development of microbial insecticides and breakthrough against foreign patent protection barrier. This study presented a systematic and in-depth study of Bt strain ST8isolated and preserved in our laboratory. Three novel cry8genes were cloned, preliminary expressed, and used for construction of gene engineered strains. The concrete results are as follows:1. This paper systematically investigated the biological characteristics of strain ST8. which was isolated from Sichuan ecological conditions. Electron microscopic observation indicated that ST8cell, with a long rod and flagellum, could form a spherical parasporal crystal. The results of the physiological and biochemical properties and H-serotype identification suggested ST8belonged to Bt serovar Londrina subspecies. SDS-PAGE results showed that ST8mainly produced about130kDa protein. Biological activity test results indicated that ST8had high insecticidal activity to H. parallela and H. ohlita.2. The cry gene-types of ST8were identified using PCR-RFLP system. The results showed that ST8harbored cry8genes. Kpn I and Dra I were further used to double digest the amplified products. The results showed that the cleaved product size of ST8amplified fragment was different from known cry8genes’, which suggested that ST8may contain potentially novel cry8genes.3. According to the amplified sequence of ST8obtained by cry8gene universal primers, two specific primers were designed in the sequence ends, respectively. And then the fragments of gene were cloned by SON-PCR technology from both sides. After splicing the known sequences, three nucleotide sequences with length about3.5kb were obtained. According to international nomenclature principles for cry gene, the three new genes were officially named as cry8Kbl, cry8Pal and cry8Qal by the international Bt insecticidal crystal protein gene nomenclature committee. cry8Kbl contained3510nucleotides encoding a protein of1170amino acids, whose predicted molecular weight was131.5kDa. cry8Pal contained3531nucleotides encoding a protein of1177amino acids, whose predicted molecular weight was132.7kDa. cry8Qalcontained3555nucleotides encoding a protein of1185amino acids, whose predicted molecular weight was132.8kDa. Structure analysis showed that, three domains of those three novel Cry8proteins located similarly, and all the three proteins had5conserved regions of insecticidal crystal protein.4. The full open reading frame sequences of the three gene were amplified with PCR primers based on their DNA sequences, respectively, and inserted into the Sac I/Not I site of E.coli-Bt shuttle expression vector pSTK to obtain the recombinant plasmid pSTK-8K, pSTK-8P and pSTK-8Q. Those recombinant plasmids were thermally shocked into SCS110for demethylation, and then transformed into the acrystalliferous mutant strain Bt HD-73-to construct Bt engineered strains, named HD8K, HD8P and HD8Q. |
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